The Novel Regulatory Role of the lncRNA–miRNA–mRNA Axis in Chronic Inflammatory Airway Diseases

Chronic inflammatory airway diseases, characterized by airway inflammation and airway remodelling, are increasing as a cause of morbidity and mortality for all age groups and races across the world. The underlying molecular mechanisms involved in chronic inflammatory airway diseases have not been fully explored. MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) have recently attracted much attention for their roles in the regulation of a variety of biological processes. A number of studies have confirmed that both lncRNAs and miRNAs can regulate the initiation and progression of chronic airway diseases by targeting mRNAs and regulating different cellular processes, such as proliferation, apoptosis, inflammation, migration, and epithelial–mesenchymal transition (EMT). Recently, accumulative evidence has shown that the novel regulatory mechanism underlying the interaction among lncRNAs, miRNAs and messenger RNAs (mRNAs) plays a critical role in the pathophysiological processes of chronic inflammatory airway diseases. In this review, we comprehensively summarized the regulatory roles of the lncRNA–miRNA–mRNA network in different cell types and their potential roles as biomarkers, indicators of comorbidities or therapeutic targets for chronic inflammatory airway diseases, particularly chronic obstructive pulmonary disease (COPD) and asthma.

INTRODUCTION
Chronic inflammatory airway diseases, including chronic obstructive pulmonary disease (COPD), bronchial asthma, bronchiectasis and cystic fibrosis (CF), remain a major problem worldwide (Soriano et al., 2020). Asthma and COPD both cause airway obstruction due to cellular and structural changes, known as airway remodelling (Aoshiba and Nagai, 2004;Hogg, 2004). Angiogenesis initiates the remodelling of the bronchial vasculature that eventually contributes to the progression of asthma and COPD (Global Strategy for Asthma Management and Prevention, 2012;Hislop, 2002; Global Initiative for Chronic Obstructive Lung Disease (GOLD), 2013). Bronchiectasis is characterized by irreversible bronchial dilatation with associated chronic cough, mucus hypersecretion, and recurrent pulmonary infections (Polverino et al., 2017). CF, a major disorder associated with bronchiectasis, is caused by mutation of a gene called cystic fibrosis transmembrane conductance regulator (CFTR), and its dysfunction results in mucus retention and chronic infection with subsequent local airway inflammation (Elborn, 2016). They are increasing as a cause of morbidity and mortality with a serious disease burden across the world. The molecular mechanisms of the pathophysiological processes involved in these diseases have not been completely interpreted. The identification of the pivotal molecules involved in chronic inflammatory airway diseases is urgently needed for exploring effective prevention measures and therapeutic strategies. In recent decades, noncoding RNAs (ncRNAs), characterized by the absence of significant proteincoding potential, have been comprehensively investigated. As two major members of the ncRNA family, long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in regulating cell differentiation (Liang and Tang, 2020), proliferation (Wang et al., 2021a), autophagy , and apoptosis . LncRNAs, a novel class of RNA transcripts with a length greater than 200 nucleotides, have been thought to have no biological function due to their lower expression levels compared with protein-coding genes, but growing evidence has confirmed that lncRNAs can regulate target gene expression from the transcriptional and posttranscriptional to posttranslational levels and play critical roles in multiple biological processes (Ransohoff et al., 2018). Unlike lncRNAs, miRNAs are a family of evolutionarily conserved ncRNAs and can bind to the 3′-untranslated region (3′-UTR) of messenger RNAs (mRNAs), leading to mRNA translation inhibition or mRNA degradation, which play a significant role in regulating cellular bioactivity Smith et al., 2022). Recently, a variety of studies have confirmed that noncoding RNAs (ncRNAs) participate in the development of chronic inflammatory airway diseases (Li et al., 2019a). Moreover, a novel regulatory mechanism among lncRNAs, miRNAs and their mRNA targets has been found in chronic inflammatory airway disease. For example, lncRNA bromodomain adjacent to zinc finger domain protein 2B (lnc-BAZ2B) promoted pulmonary inflammation and mucus secretion via BAZ2B/interferon regulatory factor 4 (IRF4) in vitro (Xia et al., 2021). The overexpression of miR-145-5p attenuated cigarette smoking extract (CSE)-stimulated apoptosis and inflammation in human bronchial epithelial cells (Li et al., 2019b). LncRNA cancer susceptibility candidate 7 (CASC7) promoted the phosphorylation of glucocorticoid receptors to increase the sensitivity of glucocorticoids in patients with severe asthma by targeting the miR-21/phosphoinositide 3kinase (PI3K)/protein kinase B (Akt) signalling pathway. This review summarizes the current progress of research on the interaction between lncRNAs and miRNAs to provide insight into their potential as new biomarkers or therapeutic targets of chronic inflammatory airway diseases.

NCRNAS IN CHRONIC INFLAMMATORY AIRWAY DISEASES
NcRNAs are commonly classified into two categories, housekeeping and regulatory ncRNAs, according to their regulatory roles. MiRNAs (transcripts between 19 and 25 nucleotides) and lncRNAs (transcripts >200 nucleotides) are major members of the regulatory ncRNA family. MiRNAs negatively regulate gene expression by target mRNA cleavage and degradation or translation inhibition, while lncRNAs negatively or positively regulate various stages of gene expression through interactions with DNA, RNA or proteins (ChewChew et al., 2018). Currently, ncRNAs are discovered by broad-range profiling methods, including bioinformatic analysis, microarrays, and next-generation sequencing, which could measure the differential expression of thousands of ncRNAs Beermann et al., 2016). Validation of the screening results can be done with low effort and relatively low costs by quantitative real-time PCR (qRT-PCR) or western blot. Human samples such as blood, urine, sputum, and lung tissues have been shown to exhibit a differentially expressed ncRNAs (Liang et al., 2019;Jakwerth et al., 2021;McDonough, et al, 2019), underscoring their potential to serve as biomarkers. In terms of the bio-sample, the blood might be better for investigating ncRNA as biomarkers in chronic inflammatory airway diseases since inflammatory processes from the lung could spill into the blood, and ncRNA obtained from blood could be representative of the inflammatory response in the lung and other tissues. In addition, it may be performed more easily than invasive methods such as lung biopsies, bronchial brushings or bronchoalveolar lavage fluid.
In addition to miRNAs, lncRNAs are also associated with the pathogenesis and development of chronic inflammatory airway diseases. For example, taurine-up-regulated gene 1 (TUG1) and metastasis-associated lung adenocarcinoma transcript 1 (Malat1) are upregulated in lung tissues of COPD patients, and their silencing inhibits the expression of fibronectin and α-smooth muscle actin (α-SMA) and improves the viability of human fetal lung fibroblast 1 (HFL1) cells following TGF-β pretreatment Hu et al., 2020). HOXA-AS2 was downregulated in lung tissues from COPD patients, and the downregulation of HOXA cluster antisense RNA 2 (HOXA-AS2) suppresses CSE-induced human pulmonary microvascular endothelial cells (HPMECs) proliferation via Notch1 signalling in vitro (Zhou et al., 2020a). Furthermore, several lncRNAs are also associated with the regulation of airway inflammation and asthma. Zhang et al.  found that brain cytoplasmic RNA 1 (BCYRN1) was upregulated in an asthma rat model and induced the proliferation and migration of airway smooth muscle cells (ASMCs) by targeting transient receptor potential canonical 1 (TrpC1). The upregulation of lncRNA transcription factor 7 (TCF7)  and lncRNA plasmacytoma variant translocation 1 (PVT1) (Austin et al., 2017) and their role in the regulation of ASMC proliferation and migration have been established in asthmatic patients targeting mitochondrial membrane domain containing 1 (TIMMDC1) and IL-6, respectively. In addition to asthma and COPD, lncRNAs also regulate CF lung disease. For example, McKiernan et al. (McKiernan et al., 2014) found that X inactivation-specific transcript (XIST) and Malat are differentially expressed in bronchial brushings of CF patients. Balloy et al. (Balloy et al., 2017) also found that several lncRNAs, such as maternally expressed gene 9 (MEG9) and bladder cancerassociated transcript 1 (BLACAT1), are downregulated in Pseudomonas aeruginosa (PA)-infected CF bronchial epithelial cells. In addition, lncRNA BGas was shown to be associated with CF by directly targeting and regulating CFTR (Saayman et al., 2016). Unfortunately, current studies on lncRNAs in bronchiectasis are limited and need further investigation. Collectively, these studies suggest that miRNAs and lncRNAs play a significant role in the pathogenesis and development of chronic inflammatory airway diseases.

LNCRNA-MIRNA INTERACTION IN CHRONIC INFLAMMATORY AIRWAY DISEASES
In addition to the regulation where both miRNAs and lncRNAs act alone on mRNAs, accumulating evidence has demonstrated that lncRNAs can interact with miRNAs to regulate mRNA expression via various posttranscriptional mechanisms. Four types of lncRNA-miRNA interactions have been demonstrated: miRNA-triggered lncRNA decay, lncRNA generating miRNA, lncRNA-miRNA competition for mRNA, and lncRNA acting as miRNA sponges/decoys (Yoon et al., 2014). Mounting studies have shown that the latter is the most common mechanism involved in various diseases, such as cancer (López-Urrutia et al., 2019), cardiovascular diseases (Huang, 2018), rheumatoid arthritis (Han et al., 2022), and cerebral diseases (Mehta et al., 2021). Under such an interaction, lncRNAs prevent miRNAs from hindering the expression of target genes, such as mRNAs, and ultimately affect the function of cells by enhancing target mRNA expression   (Figure 1). Recently, lncRNAs acting as miRNA sponges have been gradually revealed to be involved in chronic inflammatory airway diseases, such as FIGURE 1 | LncRNAs can act as a sponge for microRNAs. By binding to these microRNAs, they prevent microRNAs from binding to their target mRNAs, thereby abolishing post-transcriptional regulation. COPD and asthma. PubMed was searched using the query "lncRNAs" AND "miRNAs" AND "COPD OR asthma OR bronchiectasis OR cystic fibrosis." Articles regarding lncRNA-miRNA crosstalk discovery in chronic inflammatory airway diseases as well as in vitro and in vivo functional studies were included. They participate in the regulation of the proliferation, apoptosis, invasion, CD4 + T-cell differentiation, epithelial-mesenchymal transition (EMT), and inflammation of multiple relevant cell types in chronic inflammatory airway diseases. In the following sections, we describe the emerging roles and mechanistic functions of lncRNA-miRNA interactions in various cell types involved in chronic inflammatory airway diseases. Table 1 summarizes the crosstalk of lncRNA-miRNA-mRNA in COPD and asthma.

LncRNAs Acting as miRNA Sponges in Bronchial Epithelial Cells
Bronchial epithelial cells are the first line of defence against environmental stimuli and play a critical role in maintaining normal airway function. Dysfunction of bronchial epithelial cells results in immune homeostasis disorder, persistent inflammation, oxidative stress, and EMT (Gao et al., 2015). The involvement of lncRNA sponging miRNA in the regulation of bronchial epithelial cells in chronic inflammatory airway diseases has been established by several studies. A number of lncRNAs including myocardial-infarction-associated transcript (MIAT) (Gu et al., 2022), nicotinamide nucleotide transhydrogenaseantisense RNA1 (NNT-AS1) (Mei et al., 2020), XIST , and TUG1  are upregulated in CSEinduced human bronchial epithelial cell line (16HBE) and interact with the miR-29c-3p/HIF3A axis, the miR-582-5p/ FBXO11 axis, the miR-200c-3p/early growth response 3 (EGR3) axis, and the miR-145-5p/dual-specificity phosphatase 6 (DUSP6), respectively, to participate in CSE-induced 16HBE cell apoptosis and inflammation. Zhao et al. (Zhao et al., 2021) detected the expression of lncRNAs in the serum from patients with COPD and found that the expression of lung cancerassociated transcript 1 (LUCAT1) was significantly increased, especially in the smoker COPD group, and LUCAT1 levels were positively correlated with inflammatory cytokine (IL-1β, IL-6, and TNF-α) expression in COPD patients. Further studies in CSE-treated 16HBE cells showed that LUCAT1 acts as a miR-181a-5p sponge modulator of the expression of the downstream target genes TCF4, cyclin D1, c-Myc, and β-catenin. Silencing LUCAT1 alleviated the effects of CSE on 16HBE cell proliferation and apoptosis through the miR-181a-5p/Wnt/β-catenin pathway. The roles of Wnt/β-catenin signalling activation in cell proliferation, apoptosis, inflammation and injury repair (Zemans et al., 2011;Tanjore et al., 2013;Jang et al., 2017), as well as the potential in attenuating COPD pathogenesis, have been well demonstrated (Uhl et al., 2015). Furthermore, LUCAT1 could differentiate COPD patients from smokers and nonsmokers with high sensitivity, specificity, and accuracy. These findings suggest that LUCAT1/miR-181a-5p/Wnt/β-catenin axis may be a promising target for COPD treatment and that LUCAT1 may be a valuable indicator for differentiating COPD. Lei et al. (Lei et al., 2021) reported that in the peripheral blood samples of smokers with COPD, the expression levels of lncRNA MEG3 and miR-149-3p were negatively correlated. In the subsequent COPD model induced by CSE in vitro, they found that MEG3 expression was significantly upregulated. The knockdown of MEG3 could directly target miR-149-3p to inhibit the activation of the NF-κB signalling pathway and promote proliferation while inhibiting apoptosis of CSEtreated HBE cells, suggesting that MEG3/miR-149-3p/NF-κB axis has a crucial function in the pathogenesis of COPD. In another report from traffic-related air pollution particulate matter 2.5 (TRAPM2.5)-induced 16HBE cells, significantly elevated lncRNA RP11-86H7.1 accompanied by activation of the NF-κB signalling pathway was detected . The overexpressed RP11-86H7.1 could directly sponge miR-9-5p, enhance the expression levels of NFKB1, IL-6, IL-8, and tumor necrosis factor (TNF)-α, and promote the inflammatory response in TRAPM2.5-treated 16HBE cells, providing further insights into lncRNA-miRNA interactions regarding new treatment strategies for airway inflammatory diseases caused by PM2.5 exposure, such as COPD . The two aforementioned studies showed that the NF-κB signalling pathway regulated by lncRNA-miRNA plays a crucial role in the cell inflammatory response, proliferation and apoptosis.
Hao and his colleagues  revealed that the levels of opa interacting protein 5-antisense RNA 1 (OIP5-AS1) were elevated in smokers and smokers with COPD, and there was a negative connection between OIP5-AS1 and FEV 1 . Furthermore, OIP5-AS1 showed a talent in differentiating smokers with COPD from smokers, as revealed by the area under the curve (AUC) of 0.903. Knockdown of OIP5-AS1 reversed the effects of CSE on 16HBE cell viability and apoptosis via miR-410-3p and IL-13. All of these findings indicated that OIP5-AS1 might serve as a diagnostic biomarker as well as a promising target for COPD treatment. In addition to a regulatory role in COPD, Cai et al.  found that OIP5-AS1 expression levels increased in an in vitro asthma model (Der p1-induced BEAS-2B cells) and that OIP5-AS1, serving as the molecular sponge of miR-143-3p, promoted Der p1-induced BEAS-2B cell apoptosis and inflammation by modulating high mobility group box-1 (HMGB1), a highly conserved ubiquitous protein that has been demonstrated to be associated with the development of asthma by promoting allergen-induced airway remodelling (Hou et al., 2015). These results provide a reference for OIP5-AS1 sponging different miRNAs as therapeutic targets in asthma and COPD.
In contrast to the aforementioned lncRNAs, three reported lncRNAs showed their abilities to suppress CSE-induced 16HBE cell apoptosis and inflammation by sponging miRNAs and affecting their target genes. For example, Shen et al.  reported that small nucleolar RNA host gene 5 (SNHG5) expression was significantly decreased in COPD tissues and that high SNHG5 expression was positively correlated with FEV 1 % in patients. In CSE-treated 16HBE cells, SNHG5 expression was reduced, and miR-132 was detected as an SNHG5 miRNA target. Simultaneously, PTEN was confirmed to serve as a target gene of miR-132. SNHG5 upregulation reversed the effects of CSE on 16HBE cell proliferation, apoptosis, and inflammatory cytokine levels (IL-1β, IL-6 and TNF-α) by targeting the miR-132/PTEN axis. As described by a previous study, PTEN plays a crucial role in regulating the inflammatory response in COPD (Yanagisawa et al., 2017). These data suggest that SNHG5/miR-132/PTEN is involved in COPD progression and might serve as a potential prognostic biomarker. The other two lncRNAs, CASC2 and LOC729178, also act as miRNA sponges in COPD; their overexpression alleviated CSE-induced 16HBE cell inflammatory injury and apoptosis via the CASC2/miR-18a-5p/insuline-like growth factor I (IGF1) and LOC729178/miR-144-3p/PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) axes, respectively (Liu et al., 2021a;Wang et al., 2021b). In addition, the level of CASC2 expression was shown to be positively associated with FEV 1 (Liu et al., 2021a), which might be a promising biomarker for disease diagnosis and to have diagnostic accuracy in distinguishing COPD patients from smokers.
The abnormal airway epithelium contributes to both CF and bronchiectasis (Peng et al., 2020;Chen et al., 2018;Barbry et al., 2020). Insufficient growth and differentiation of epithelial cells can lead to persistent mucosal injury that promotes bacterial colonization and neutrophilic inflammation. There has been evidence that the lncRNAs MEG9 and BLACAT1 are downregulated in CF bronchial epithelial cells infected with PA (Balloy et al., 2017). MiR-145-5p and miR-494 are directly involved in the dysregulation of CFTR in bronchial epithelial cells, as mentioned above (Oglesby et al., 2013). Further research is needed to investigate whether the lncRNA-miRNA-mRNA axis affects bronchial epithelial cells in CF and bronchiectasis, which may present novel therapeutic opportunities.

LncRNAs Act as miRNA Sponges in Pulmonary Microvascular Endothelial Cells
Angiogenesis and microvascular remodelling are known features of chronic inflammatory airway diseases, such as COPD and asthma (Alagappan et al., 2013;Bakakos et al., 2016). Cigarette smoking can initiate pulmonary vascular impairment by directly injuring endothelial cells, resulting in the enhancement of cell apoptosis and vascular permeability, reduced epithelial barrier formation, angiogenesis and high levels of oxidative stress, thereby contributing to COPD development (Alagappan et al., 2013;Long et al., 2018). A number of lncRNAs have been thought to be promising for the treatment of COPD by regulating pulmonary microvascular endothelial cell function through targeting the miRNA/mRNA axis. A recent report demonstrated the downregulation of LINC00612 in COPD tissues and in human pulmonary microvascular endothelial cells (HPMECs) exposed to CSE . The upregulation of LINC00612-inhibited cell apoptosis, inflammation, and oxidative stress in HPMECs induced by CSE positively regulated Notch signalling by sponging miR-31-5p, indicating that LINC00612/miR-31-5p/Notch axis could function as a potential biomarker and treatment target for COPD in the future. Interestingly, the Notch signalling pathway has been acknowledged to play a significant role in asthma (Kang et al., 2009;Gu et al., 2012). Therefore, whether LINC00612/miR-31-5p/Notch can be also a therapeutic target for asthma treatment needs to be explored in the future. Song et al. (Song et al., 2020) found increased expression of MIR155 host gene (MIR155HG) in lung tissues of smokers with COPD compared with controls with COPD and CSE-treated HPMECs. In CSE-treated HPMECs, MIR155HG acts as a sponge for miR-128-5p, and luciferase reporter assays subsequently showed an interaction between miR-128-5p and BRD4. BRD4 has been revealed to play a vital role in the regulation of inflammation, oxidative stress, and innate immunity in COPD patients (Devaiah et al., 2016;Malhotra et al., 2017;Tang et al., 2019). Elevated MIR155HG expression promoted HPMEC apoptosis and inflammation in vitro through the miR-128-5p/BRD4 regulatory axis. MIR155HG deletion using small interfering RNAs (siRNAs) in vitro suppressed apoptosis and inflammation in CSE-treated HPMECs, indicating therapeutic potential (Fehrenbach et al., 2017). In addition, Li et al. (Li et al., 2019b) found that MIR155HG regulated M1/M2 macrophage polarization in COPD by regulating IL-1β, IL-10, IL-12, and TNF-α expression, suggesting that MIR155HG might be involved in the development of COPD. Therefore, more studies are needed to confirm whether MIR155HG can regulate macrophage polarization by sponging miRNA, providing a novel treatment strategy for COPD. Chen et al. (Chen et al., 2021b) investigated the function of TUG1 in an in vitro COPD model with CSE-treated HPMECs and showed that overexpression led to apoptosis in HPMECs by modulating the miR-9a-5p/Bcl-2-like protein 11 (BCL2L11) axis, suggesting that TUG1/miR-9a-5p/BCL2L11 is a potential effective target for COPD treatment.
Despite the crucial role of angiogenesis and microvascular remodelling in asthma, studies on the role of lncRNA-miRNA crosstalk in the regulation of HPMECs function contributing to the development of asthma are rare, which requires further exploration.

LncRNAs Acting as miRNA Sponges in T Lymphocytes
Lymphocytes are the main immune cells mediating airway inflammation in COPD and asthma. T lymphocytes have been shown to play a central role in the pathophysiology of asthma. T lymphocytes are generally divided into two major types: CD4+T cells and CD8+T cells. CD4 + T cells initiate inflammatory and allergic responses in allergic asthma, leading to two asthma phenotypes: Th2 and non-Th2 (Sze et al., 2020). Th2-asthma (i.e., eosinophilic asthma) is well established to play a leading role in asthma development as more than half of asthma cases have a Th2 phenotype, characterized by the secretion of high levels of IL-4, IL-5, and IL-13 by Th2 cells (Masuda et al., 2008;Hammad and Lambrecht, 2021). Non-Th2 asthma (i.e., neutrophilic asthma), by contrast, is characterized by the secretion of high levels of interferon-gamma (IFN-γ) and IL-17 by Th1 and Th17 cells (Sze et al., 2020). Biased differentiation of CD4 + T cells contributes to abnormal inflammation in asthma (Wu et al., 2016). LncRNAs and miRNAs alone have been shown to play regulatory roles in asthma by regulating CD4 + T-cell function (Wang et al., 2021c). Notably, three studies observed a novel regulatory role of lncRNAs targeting miRNAs in regulating the Th1/Th2 balance (Liang and Tang, 2020;Wei et al., 2020;Sun et al., 2021). For example, Sun et al.  observed that TUG1 is differentially expressed in the monocytes of asthmatic children. In further in vitro experiments on macrophages treated with house dust mites, they demonstrated that TUG1 could regulate B7-H3 expression in macrophages by sponging miR-29c, which then controls Th2 differentiation, providing novel potential diagnostic biomarkers and therapeutic targets for asthma. Liang et al. (Liang and Tang, 2020) demonstrated that MALAT1 could sponge miR-155, affecting the Th1/Th2 balance through a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)-dependent mechanism, which might aid in the development of therapies for ameliorating inflammation in asthma. Wei et al. (Wei et al., 2020) also investigated the function of PVT1, showing that overexpression significantly decreased the Th1/Th2 ratio in CD4 + T cells by depressing miR-15a-5p expression and therefore induced PI3K-Akt-mTOR signalling. Previous studies found that PI3K/Akt activation facilitated the differentiation of Th cells into Th2-like CD4 + T cells (Ward and Peter, 2003;Arimura et al., 2004). Therefore, the lncRNA PVT1/miR-15a-5p/PI3K-Akt-mTOR axis was implicated in asthma development by promoting Th1/Th2 imbalance.
In addition to the aforementioned regulatory role of the lncRNA-miRNA axis in Th1/Th2 imbalance, Qiu et al. (Qiu et al., 2019) found that the lncRNA MEG3 plays a role in the regulatory T-cell (Treg)/Th17 balance by sponging target miRNAs. They found that MEG3 expression levels were upregulated and negatively associated with miRNA-17 expression in CD4 + T cells of patients with severe asthma. Silencing of MEG3 expression using siRNA in CD4 + T cells reduced the expression of the Th17 transcription factors orphan receptor (ROR)γt and IL-17. Additionally, miR-17 was detected as a target of MEG3, and it could suppress the Th17 response or promote it by targeting RORyt, suggesting that MEG3/miRNA-17/RORyt axis plays a role in the Th17 imbalance in asthma. Previous studies found that the frequency of Th17 cells was markedly increased in patients with moderate to severe asthma and that Treg/Th17 imbalance is more closely related to asthma progression and severity than Th1/Th2 imbalance (Shi et al., 2011;Wang et al., 2018b). Therefore, we can assume that MEG3/miRNA-17/RORyt axis could be a biomarker of asthma severity. However, Feng et al. (Feng et al., 2020) found that MEG3 was downregulated in the serum of patients with neutrophilic asthma compared with controls, which contradicts the above finding and requires further research and validation.
Although the critical role of CD4+T cells in the physiopathology is indisputable, recent evidence suggests that CD8+T cells may also be involved in asthma. Den et al. (den Otter et al., 2016) found that the decline of FEV 1 both at baseline and follow-up correlated with the number of CD8+T cells in asthmatic airways, hinting a positive correlation between the disease severity and CD8+T cells. Tsitsiou et al. (Tsitsiou et al., 2012), using transcriptome analysis, showed that severe asthma is associated with the activation of circulating CD8 + T cells but not CD4 + T cells, which is correlated with the changes in the expression of miR-146a/b and miR-28-5p as well as 167 different lncRNAs that might regulate CD8 + T-cell function. Therefore, the regulatory roles of lncRNAs or miRNAs or lncRNA-miRNA interactions in CD8 + T-cell function merit further exploration. If possible, this might provide a novel therapeutic approach to the treatment of severe asthma.
As with asthma, Th17-mediated pulmonary inflammation is also crucial to COPD (Ponce-Gallegos et al., 2017). In a COPD mouse model, He et al. (He et al., 2021) reported that upregulated miRNA-21 could promote Th17 cell differentiation, contributing to increased levels of inflammatory cytokines via the Smad7/ TGF-β pathway. However, the role of lncRNA-miRNA-mRNA axis in COPD through regulating T cells has rarely been studied, which needs to be investigated.

LncRNAs Act as miRNA Sponges in Airway Smooth Muscle Cells
Airway remodelling is an important feature of chronic inflammatory airway disease, especially COPD and asthma. An important contribution to airway remodelling and irreversible bronchoconstriction is increased airway smooth muscle (ASM) mass, which is thought to be brought on by airway smooth muscle cell (ASMC) proliferation, migration and hypertrophy (Salter et al., 2017). The role of lncRNA-miRNA-mRNA regulatory network in the asthmatic process by modulating platelet-derived growth factor (PDGF)-BB-stimulated ASMCs has been increasingly reported in recent years. For example, lncRNA growth arrestspecific transcript 5 (GAS5), lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), and lncRNA PVT1 can act as "sponges" of miR-10a, miR-9-5p, and miR-29c-3p, respectively, and are involved in the biological processes of ASMC proliferation, migration, apoptosis and inflammation in asthmatic patients (Zhang et al., 2018b;Wei et al., 2020;Wang et al., 2021d). TUG1, serving as the molecular sponge of miR-138-5p, miR-590-5p, miR-216a-3p, and miR-181b, has been confirmed to be involved in ASMC proliferation and migration in human asthma by modulating E2F transcription factor 3 (E2F3), fibroblast growth factor 1 (FGF1), Smurf2 and HMGB1, respectively (Lin et al., 2019a;Huang et al., 2021;Zhou et al., 2021). Similarly, the lncRNA Malat1 also contains multiple binding sites to miRNAs and sponges miR-150 (Lin et al., 2019b) and miR-133a  in addition to the aforementioned miR-155 (Liang and Tang, 2020), thereby regulating PDGF-BB-stimulated ASMC proliferation and migration. These findings provide a theoretical basis for lncRNAs-miRNAs-mRNAs axis being potential therapeutic targets for asthma.
Multiple signalling pathways are involved in the pathogenesis of asthma by regulating airway smooth muscle cell function. Liu et al. (Liu et al., 2020) found that lncRNA CASC7 inhibits the PI3K/Akt signalling pathway by targeting miR-21 and promotes the phosphorylation of glucocorticoid receptors to increase the sensitivity of glucocorticoids in patients with severe asthma. Indeed, miR-21 overexpression could lead to steroid-insensitive airway inflammation and airway hyperresponsiveness (AHR) . These findings indicated the therapeutic potential of CASC7/miR-21/PI3K/Akt axis in AHR. Yu et al. discovered that lncRNA H19 overexpression reduced the growth and migration of ASMCs by acting on the miR-21/PTEN/Akt pathway . Interestingly, several studies with an asthmatic mouse model and in asthmatic children indicate that the upregulation of miR-21 promotes increased expression of Th2 cytokines and inhibits Th1 cytokine expression (Lu et al., 2009;Elbehidy et al., 2016;Hammad Mahmoud Hammad et al., 2018). Therefore, lncRNA CASC7/ miR-21/PI3K/Akt axis and H19/miR-21/PTEN/Akt axis are potential therapeutic targets for asthma, and whether they can regulate T-cell differentiation to treat asthma needs further study. Gao et al. (Gao et al., 2021) found that LINC p53induced transcript (PINT) overexpression decreased the serum levels of IgE and AHR, airway inflammation, and pathological changes in the lung in an asthma rat model. In vitro, LINC-PINT overexpression retarded abnormal ASMC growth to attenuate the progression of asthma by regulating the miR-26a-5p/PTEN axis. PTEN is a phosphatase that acts as a negative regulator of PI3K/AKT signalling, and the PTEN/PI3K/AKT signalling pathway plays multifaceted roles in asthma pathogenesis (Yoo et al., 2017). Furthermore, the PI3K/AKT/mTOR pathway was also involved in the regulation of AMSC proliferation and migration by lncRNA RP5-857K21.7 sponging miR-508-3p .
In addition to the PI3K/AKT/mTOR pathway, the janus kinase (JAK) signalling pathway has been shown to play a key role in asthma KimHye et al., 2021). Shen et al.  showed that lncRNA FTX could inhibit the proliferation and migration of ASMCs caused by PDGF-BB by targeting miR-590-5p and that JAK2 was a direct target of the FTX/miR-590-5p signalling axis, the overexpression of which reversed the inhibitory effect on proliferation and migration and the apoptosis-inducing effect of miR-590-5p in ASMCs, highlighting the crucial regulatory role of the FTX/ miR-590-5p/JAK2 axis in ASMC proliferation, migration, and apoptosis.
Liu et al.  demonstrated that LNC00882 promotes the proliferation of ASMCs by enhancing Wnt/βcatenin signalling by sponging microRNA-3619-5p, supporting a role of LNC00882/microRNA-3619-5p/Wnt/β-catenin in airway remodelling. Another study from Perry et al. (Perry et al., 2014) found that LINC00882 is complementary for miR-371 (a total of 7 sites) through microarray and quantitative real-time PCR. MiR-371 has been confirmed to have the ability to inhibit Runt-related transcription factor 3 (Runx3), a transcription factor that promotes Th1 differentiation, by combining the other four miRNAs, thereby modulating the Th1/Th2 balance in asthma (Qiu et al., 2017). Whether LNC00882 modulates the Th1/Th2 balance by binding to miR-371 remains to be investigated.
Airway smooth muscle cells act as the main effector cells and their proliferation represents a major characteristic of airway remodelling in COPD. However, few studies explore the role of lncRNA-miRNA-mRNA axis in COPD by regulating ASMCs function, which needs to be confirmed further.

LncRNAs Act as miRNA Sponges in Other Pulmonary Cell Types
The key features of chronic inflammatory airway disease pathophysiology are small airway fibrosis, structural damage, and extracellular matrix deposition resulting in remodelling of the airways (Ma et al., 2020;Mehrad et al., 2012;Rout-Pitt et al., 2018). Airway remodelling in patients with these diseases is related to EMT, in which fibroblasts and airway epithelial cells are the two central effector cells involved Sohal et al., 2010). It has been shown that the MIAT/miR-29c-3p/ HIF3A and TUG1/miR-145-5p/DUSP6 axis are integral in promoting EMT and collagen deposition among lung fibroblast through upregulation of 3b1;-SMA, vimentin and collagen I levels (Gu et al., 2022). They also promote apoptosis and inflammation induced in CSE. Additionally, MIAT/miR-29c-3p/HIF3A appear to have a regulatory role in CSE-induced inflammation in the murine type II alveolar epithelial cell line MLE12 (Gu et al., 2022).
Furthermore, other immune cells, such as macrophages and neutrophils, also contribute to the inflammatory and immune process responsible for much of the pathology found in patients with chronic inflammatory airway disease. Previous studies have shown that ncRNAs act as crucial regulators in these cells. Shi (Shi et al., 2020), Pei. et al. (Pei et al., 2020) found that ncRNAs such as AK085865, MIR155HG, miR-142-5p, and miR-130a-3p could affect macrophage polarization in the lungs, as well as related airway inflammation and airway remodelling in COPD and asthma. Tazi et al. (Tazi et al., 2016) found that elevated miRc1/miR17-92 cluster expression negatively regulated autophagy and CFTR function in CF macrophages. To date, only a few studies have explored the role of ncRNA in neutrophils. Neutrophil-derived miRNA-223-3p has been shown to modulate Toll-like receptors (TLRs)/Th17 and the endoplasmic reticulum stress response in asthmatic sputum (Gomez et al., 2020). Moreover, MiR-636 was found to increase the neutrophils of CF patients' blood and might serve as a new biomarker (Bardin et al., 2019). Currently, studies that explore the role of the lncRNA-miRNA-mRNA axis in these cells are lacking, and they ought to be encouraged

CLINICAL IMPLICATIONS IN CHRONIC INFLAMMATORY AIRWAY DISEASES
In recent years, a growing number of studies have demonstrated that crosstalk among lncRNAs, miRNAs and mRNAs is involved in the pathophysiology of chronic inflammatory airway diseases. Recent studies have focused on lncRNA-miRNA crosstalk as a possible biomarker of COPD and asthma. While the axis influences many biological processes in COPD, including diagnosis and treatment, research into asthma attempts to identify phenotypes and predict therapeutic responses.

LncRNA-miRNA-mRNA Axis in COPD
Thirteen lncRNAs are implicated in the physio-pathological process of COPD by sponging miRNAs (Figure 2). Of these, eleven differentially expressed lncRNAs have been found in human biosamples (including whole blood, serum, sputum, and lung tissue samples) from COPD patients: NNT-AS1 (Mei et al., 2020), MEG3 (Lei et al., 2021), LUCAT1 (Zhao et al., 2021), SNHG5 , CASC2 (Liu et al., 2021a), TUG1 , OIP5-AS1 , LOC729178 , XIST , LINC00612 , and MIR155HG (Song et al., 2020). There is a possibility that these lncRNAs could be clinically helpful in diagnosing and treating COPD. Compared to smokers without COPD, LUCAT1, CASC2, and OIP5-AS1 show a more sensitive expression pattern, thus enabling a helpful distinction between smokers with and without COPD. There is also evidence that LUCAT1 expression correlates with inflammation in COPD patients and that TUG1, SNHG5, CASC2, and OIP5-AS1 expression levels correlate with the severity of airflow limitation, supporting their roles as potential biomarkers for COPD progression. Moreover, TUG1 may also be a novel and promising biomarker for COPD complicated with PAH. Wang et al.  previously demonstrated that TUG1 was significantly upregulated in patients with PAH and that TUG1 knockdown significantly prevented the development of PAH in vivo. This may provide multiple opportunities to further investigate a role of the lncRNA-miRNA-mRNA axis in COPD complicated with PAH.
Smoking and aberrant epithelial responses are risk factors for COPD and lung cancer (Murray et al., 2017). A large proportion of lung cancer patients showed a history of COPD, and the possible link between COPD and lung cancer has been demonstrated (Ytterstad et al., 2016;Hou et al., 2019). The blood levels of MEG3 are higher in COPD patients but lower in non-small-cell lung cancer patients Lv et al.,2021). This contradictory finding requires further research and validation.

LncRNA-miRNA-mRNA Axis in Asthma
Thirteen lncRNAs sponging various miRNAs play critical roles in airway epithelial cells, smooth muscle cells, and T cells in asthma FIGURE 3 | The lncRNA-miRNA crosstalk is involved in the pathogenesis of asthma by regulating CD4 + T cells and airway smooth muscle cells. Different lncRNAs regulate different miRNAs through the sponge effect, and then inhibit Mrna. The dotted boxes in the picture show typical lncRNA-miRNA interactions that function in asthma, which correspond to the cellular process they act on through pink arrows. DC: Dendritic cell; ASMCs: airway smooth muscle cells.
Frontiers in Molecular Biosciences | www.frontiersin.org June 2022 | Volume 9 | Article 927549 ( Figure 3). MEG3 (Qiu et al., 2019), TUG1 , Malat1 (Liang and Tang, 2020), CASC7 , and H19  showed differential expression in asthmatic patients. Various asthma phenotypes might be treated differently in terms of clinical management. For example, inhaled corticosteroids (ICS) are less effective in treating neutrophilic asthma, particularly in patients with severe ICS resistance (Heffler et al., 2018). A key component of precision medicine is the identification of accurate biomarkers that can be used to distinguish different disease phenotypes. These biomarkers can help determine the optimal treatment for each patient. Various lncRNA-miRNA-mRNA axes are associated with asthma severity or inflammatory phenotypes. TUG1 and Malat1 promote Th2 asthma through the TUG1/miR-29c/B7-H3 axis and Malat1/miR-155/CTLA4 axis, respectively, whereas MEG3 promotes non-Th2 asthma through the MEG3/miR-17/RORγt axis. In addition, the lncRNA CASC7/miR-21/PI3K/Akt axis may be beneficial for alleviating AHR in asthmatic patients by increasing sensitivity to glucocorticoids.

LncRNA-miRNA-mRNA Axis in Other Chronic Inflammatory Airway Diseases
In addition to COPD and asthma, CF and bronchiectasis are usually defined as chronic inflammatory airway diseases. Chronic infection, inflammation, mucus hypersecretion, and bronchiectasis are their common features. As we described before, there is now growing literature to suggest that ncRNAs may be novel therapeutic targets for CF via the regulation of inflammation and mucus synthesis or directly/indirectly target CFTR. Notably, lncRNA MEG9 and lncRNA BLACAT1 were found to be significantly downregulated in PA-infected CF bronchial epithelial cells (Balloy et al., 2017). Interestingly, Huang et al.  found that differentially expressed miR-223-3p and miR-92b-5p were associated with PA colonization in bronchiectasis patients, and their expression levels were significantly correlated with sputum inflammatory biomarker levels (IL-β and IL-8), suggesting that these miRNAs could be biomarkers associated with PA colonization.
Although current studies on the lncRNA-miRNA-mRNA axis in CF and bronchiectasis are limited, these findings provide clues for it being a therapeutic target to mitigate the adverse impacts caused by PA colonization in CF or bronchiectasis. Therefore, more investigations either in vivo or in vitro are urgently needed on this topic to better recognize the role of the lncRNA-miRNA-mRNA axis in chronic inflammatory airway diseases.

CONCLUSION AND PERSPECTIVES
In recent years, a growing number of studies confirm that lncRNAs and miRNAs have multifaceted and cell-type-specific functions, and might be involved in the pathogenesis and development of chronic inflammatory airway diseases, particularly COPD and asthma. This review sheds light on the expression profiles of lncRNA-miRNA-mRNA axis in various cell types involved in the pathophysiology of chronic inflammatory airway diseases and their potential function as diagnostic biomarkers and therapeutic targets for COPD and asthma via the regulation of cell proliferation, migration, differentiation, apoptosis, and inflammation.
Notably, the crosstalk of lncRNA and miRNA has excellent expectations regarding their potential as biomarkers for COPD diagnosis and complications, identifying asthma phenotypes and predicting therapeutic responses to glucocorticoids. Clinically, COPD and asthma are chronic airway inflammatory diseases with different pathogenesis but some overlaps. COPD and asthma involve airway inflammation and airway remodelling mediated by epithelial cells, T lymphocytes, endothelial cells, and airway smooth muscle cells. And several patients have both COPD and asthma features clinically. MEG3, OIP5-AS1, and TUG1 are involved in asthma and COPD pathogenesis by sponging different miRNAs. However, few studies have found that the lncRNA-miRNA-mRNA axis is associated with both COPD and asthma. Further research into ncRNA and ncRNA axis as biomarkers for asthma overlap COPD is warranted, which may contribute to asthma overlap COPD treatment.
CF and bronchiectasis are both characterized by chronic infection, mucus hypersecretion, and bronchiectasis. To date, the lncRNA-miRNA-mRNA axis has not been systematically studied concerning CF and bronchiectasis. Several ncRNAs, such as MEG9, BLACAT1, miRc1/miR17-92, miR-223-3p, miR-92b-5p and miR-636, have been found to be associated with airway epithelial cells, neutrophils, or macrophages in CF and bronchiectasis. Further research is required into the role of the lncRNA/miRNA/mRNA axis in CF and bronchiectasis.
There is still a long way to go to explore the functions and mechanisms of lncRNA-miRNA crosstalk. Firstly, although studies on the lncRNA-miRNA-mRNA axis have widely used mature detection technologies, including microarrays, RNA-sequencing, qRT-PCR, western blot, luciferase reporter assays, and RNA pulldown experiments, the reliability of detection results remains to be confirmed, as it may be affected by the technology types, sample preparation and storage, and the in vitro environment. Secondly, ncRNAs seem to be stable in human biofluids while some studies have raised concerns about sample quality, storage time and temperature that may affect the lncRNA-miRNA-mRNA network (Lorenzen and Thum, 2012;Jalali et al., 2013;Lorenzen et al., 2015;Weckbach et al., 2016;Glinge et al., 2017), which need further investigation. Thirdly, most published studies focus on the "sponge" effect between lncRNA and miRNA, the other lncRNA functionalities such as decoy, guide and scaffold affecting chronic inflammatory airway diseases need to be further studied, which may provide novel therapeutic implications. Finally, several challenges also need to be addressed before implementing lncRNA-miRNA-mRNA axes in clinical settings, including cell/tissue-specific delivery, binding affinity, stability, and sophisticated screening technologies.
In conclusion, there is no doubt that a better understanding of the crosstalk between lncRNAs and miRNAs will provide better strategies for disease diagnosis and treatment and that ncRNA networks will emerge as promising biomarkers and therapeutic targets of chronic inflammatory airway diseases, especially COPD and asthma in the near future.


s2fieldsofstudy: Biology Chemistry

text:
Lipoproteins Cause Bone Resorption in a Mouse Model of Staphylococcus aureus Septic Arthritis

Septic arthritis, most often caused by Staphylococcus aureus, is a rapidly progressive and destructive joint disease with substantial mortality and morbidity. Staphylococcus aureus lipoproteins (Lpps) are known to induce arthritis and bone destruction. Here, we aimed to investigate the bone resorptive effect of S. aureus Lpps in a murine arthritis model by intra-articular injection of purified S. aureus Lpps, synthetic lipopeptides, and live S. aureus strains. Analyses of the bone mineral density (BMD) of the distal femur bone were performed. Intra-articular injection of both live S. aureus and purified S. aureus Lpps were shown to significantly decrease total- and trabecular BMD. Liquid chromatography–mass spectrometry analyses revealed that the Lpps expressed by S. aureus SA113 strain contain both diacyl and triacyl lipid moieties. Interestingly, synthetic diacylated lipopeptide, Pam2CSK4, was more potent in inducing bone resorption than synthetic triacylated lipopeptide, Pam3CSK4. Modified lipoproteins lacking the lipid moiety were deprived of their bone resorptive abilities. Monocyte depletion by clodronate liposomes fully abrogated the bone resorptive capacity of S. aureus lipoproteins. Our data suggest that S. aureus Lpps induce bone resorption in locally-induced murine arthritis, an effect mediated by their lipid-moiety through monocytes/macrophages.

INTRODUCTION
Septic arthritis remains one of the most dangerous joint diseases due to its rapidly progressive and destructive nature (Sharff et al., 2013). The most common pathogen associated with bacterial arthritis is Staphylococcus aureus (Kaandorp et al., 1997). Despite advances in the use of antibiotics, up to 50% of patients experience permanent joint dysfunction (Jin et al., 2021). Septic arthritis might be associated with certain comorbidities such as osteoporosis, March 2022 | Volume 13 | Article 843799 Schultz et al.
Staphylococcal Lipoproteins Cause Bone Resorption as in a mouse model septic arthritis caused by intravenous (i.v.) injection of S. aureus leads to a rapid systemic bone resorption (Verdrengh et al., 2006). Increasing evidence has demonstrated bacterial components cause joint inflammation and bone destruction. Antibiotickilled S. aureus is known to induce synovitis and destructive arthritis, which is triggered by the bacterial cell wall debris (Ali et al., 2015c). The lipoproteins (Lpps) contained in the cell wall of S. aureus consist of a protein-and a lipidmoiety (Nguyen et al., 2020). The activation of pattern recognition receptors, such as Toll-like receptors (TLRs), is stimulated by the lipid-moiety of S. aureus Lpps which consequently initiates a strong innate immune system response (Nguyen and Gotz, 2016). Lately, S. aureus Lpp has been shown to play an important role in various infectious or inflammatory in vivo settings (Mohammad et al., 2019(Mohammad et al., , 2020Kopparapu et al., 2021). In a local murine knee joint model (Mohammad et al., 2019), the initiated innate immune response has a profound impact on the inflammation and destruction of joints. This effect is primarily mediated by the recruitment of monocytes/macrophages (Mohammad et al., 2019), which are the cells that can differentiate into osteoclasts and induce bone resorption (Boyle et al., 2003). An imbalance in the bone remodeling process, caused by an excess of osteoclasts and a deficit of osteoblasts, can induce osteoporosis (Fischer and Haffner-Luntzer, 2021). Currently the golden standard used to determine osteoporosis is measurement of bone mineral density (BMD). Trabecular composition is known to determine the strength of bone and consequently impacts several skeletal conditions (Adams, 1992). Osteoclasts and osteoblasts which are responsible for maintaining bone homeostasis, express a variety of TLRs. Bacterial Lpps, ligands to TLRs, can therefore affect the differentiation and activation of osteoclasts and osteoblasts. Staphylococcus aureus produces both diacylated and triacylated Lpps depending on the environment (Kurokawa et al., 2009(Kurokawa et al., , 2012. Diacylated and triacylated Lpps induce signal transduction through the TLR2/TLR6 and TLR2/TLR1 heterodimers, respectively (Jin et al., 2007;Kang et al., 2009). The synthetic lipopeptides, Pam 2 CSK 4 (Pam2Cys) and Pam 3 CSK 4 (Pam3Cys), imitate the lipid-moiety portion of diacylated and triacylated bacterial Lpps, respectively, and can therefore induce the same signaling response as original structures of Lpps (Kang et al., 2009). We hypothesize that S. aureus Lpp is one of the driving forces for bone resorption in septic arthritis.
In the present study, we used the murine septic arthritis model to investigate bone resorption induced by S. aureus Lpps in septic arthritis. In accordance with our hypothesis, both the live S. aureus LS-1 strain and the purified S. aureus Lpps, induced BMD loss, in a lipid-moiety dependent manner. We found that the Lpps expressed by S. aureus SA113 strain contain both diacyl and triacyl lipid moieties. The diacylated lipid-moiety of the synthetic lipopeptide Pam2Cys was more potent in inducing bone resorption than the triacylated lipidmoiety, Pam3Cys. Finally, monocytes, and not neutrophils, were determined to be the culprits behind S. aureus Lpp-induced bone loss.

Mice
Eight-to 12-week-old female NMRI mice were purchased from Envigo (Venray, Netherlands) and stored under standard temperature, light, and nutrition conditions in the animal facility of the Department of Rheumatology and Inflammation Research, University of Gothenburg.

Preparation of Bacterial Solutions
The S. aureus LS-1 (Bremell et al., 1991) and S. aureus Newman strains were prepared as described. The bacterial strains were stored at a temperature of −70°C. Upon use, the solution was thawed, washed with sterile phosphate-buffered saline (PBS), and adjusted to the concentration of 1 × 10 4 colony-forming units (CFU)/knee for the LS-1 strain experiments or into a concentration of 4 × 10 3 CFU/knee for the Newman strain experiments.
Expression and Purification of Lpl1(+sp) and Lpl1(−sp) Staphylococcus aureus lipoproteins Lpl1(+sp) and Lpl1(−sp) were prepared and purified, as previously described (Nguyen et al., 2015). Lpl1(+sp) and Lpl(−sp) were extracted from the membrane fraction of S. aureus SA113 (pTX30::lpl1-his) and cytoplasmic fraction of S. aureus SA113Δlgt (pTX30::lpl1(− sp)-his) respectively, as previously explained (Mohammad et al., 2019). Furthermore, SDS-PAGE was used to confirm the Lpl1-his purification, as conducted previously (Nguyen et al., 2015). A freezer was prepared to a temperature of −70°C to store the purified compounds of Lpl1(+sp) and Lpl1(−sp) until usage. Subsequently, before each experiment the compounds were diluted in sterile PBS to a concentration of 10 μg/knee that is known to be able to induce synovitis in mice if injected intraarticularly (Mohammad et al., 2019).
Frontiers in Microbiology | www.frontiersin.org or PBS; (4) synthetic lipopeptides, Pam 2 CSK 4 and Pam 3 CSK 4 (EMC, Tübingen, Germany), or PBS; (5) purified Lpl1(+sp) or PBS in monocyte/macrophage-and neutrophil depleted mice; or (6) purified Lpl1(+sp) in mice treated with TNF-α inhibitor, IL-1 receptor antagonist, or PBS as a treatment control group. The diameter of the knee joints of mice was measured regularly with a caliper to determine the severity of the induced arthritis.

In vivo Cell Depletion Procedures
The selective elimination of macrophages can be obtained through the use of clodronate liposomes (Liposoma BV, Netherlands;Van Rooijen and Sanders, 1994). To deplete both synovial residual macrophages as well as systemic monocytes, NMRI mice were i.a. injected with 20 μl of clodronate liposomes into the knee joints followed by intravenous injection with 200 μl of clodronate liposomes (Liposoma BV, Netherlands), respectively. The i.a. injection for the local depletion was performed 1 day prior to exposing the knee joints with either Lpl1(+sp) or PBS, while the intravenous injections for the systemic depletion were performed on the day before the experiment, and continued onto days 1, 3, and 5 of the experiment after exposure to either Lpl1(+sp) or PBS. In the same manner, PBS control liposomes (Liposoma BV, Netherlands) were injected into another set of mice in order to serve as controls, as previously described (Mohammad et al., 2019).
For the selective depletion of murine blood neutrophils, the monoclonal antibody Anti-Ly6G (clone 1A8; BioXCell) was used (Daley et al., 2008). A dose of 400 μg of anti-Ly6G or isotype control (clone 2A3; BioXCell) in 200 μl of PBS/mouse was injected intraperitoneally into NMRI mice to deplete the mice of systemic neutrophils, or to serve as controls, respectively. The injections were performed 1 day prior to exposing the knee joints with either Lpl1(+sp) or PBS, and continued onto days 1 and 4 after exposure to either Lpl1(+sp) or PBS, as previously explained (Mohammad et al., 2019).

Treatment With TNF-α Inhibitor or IL-1 Receptor Antagonist in NMRI Mice in S. aureus Lpp-Induced Bone Loss
The soluble TNF receptor, Etanercept (Enbrel; Wyeth Europa), which is known to fully inhibit the biological function of murine TNF (Fei et al., 2011;Ali et al., 2015b), was used in the experiment as anti-TNF treatment. Subcutaneous (s.c.) injections of Etanercept (0.2 mg/mouse in 0.1 ml of PBS) were performed. The administration began 2 days prior to exposing the knee joints with Lpl1(+sp), and continued on the day the knee joints where i.a. injected with Lpl1(+sp), and were followed by every other day until the termination date. PBS was s.c. injected as control treatment on days when Etanercept was not administered.
To block the function of IL-1 in the murine model, the IL-1 receptor antagonist Anakinra (Kineret; Amgen) was used as anti-IL-1 treatment (Sgroi et al., 2011;Ali et al., 2015a). Anakinra (1 mg/mouse in 0.1 ml of PBS) was s.c. administered daily, starting 2 days prior to exposing the knee joints with Lpl1(+sp), and continued until the termination date.
Phosphate-buffered saline was administered s.c. daily in the control treatment group, starting 2 days prior to exposing the knee joints with Lpl1(+sp), and continued until termination day.

Microcomputed Tomography
Following the termination date of the experiments, the mice knee joints were suspended in 4% formaldehyde for at least 7 days. To detect induced bone loss, mice knee joints were transferred to PBS and subsequently scanned ex vivo in the Skyscan1176 microcomputed tomography (micro-CT) scanner (Bruker, Antwerp, Belgium). During scanning, the voltage and current were set to 55 kV and 455 μA, respectively, with a voxel size of 9 μm and an aluminum filter of 0.2 mm and an exposure time of 755 ms. The scanning angular rotation was set to 180°, and the X-ray projections were retrieved at 0.42° intervals.
To obtain the BMD of mice knee joints, a morphometric analysis was performed on the distal femur bone. A 0.86 mm section of the distal femur was obtained from 100 micro-CT image slices, 50 slices above and below the reference point, for this experiment the growth plate was selected to be the reference point. The morphometric analysis was performed with the CT-Analyzer as previously described (Fatima et al., 2017).
For chromatographic separation, a 1,100 series from Agilent Technologies (Santa Clara, CA, United States) was employed. The system consists of an autosampler (G1329A), a binary pump (G1312A), a degasser (G1316A), and a column compartment (G1316A). Instrument control was established by ChemStation. For sample analysis, an Xselect CSH C18 (100 × 2.1, 3.5 μm, 130 Å) from Waters™ Corporation (Milford, MA, United States) was used at 40°C with a flow rate of 0.3 ml/min. Mobile phase A consisted of 0.05% aqueous TFA and mobile phase B was composed of 0.05% TFA in ACN/ IPA 1:1. The separation started at 25% B for 0.5 min, followed by an increase to 95% B in 6.5 min. To elute all the analytes and clean the column, 95% B was maintained for 20 min. After a steep decrease to 25% B in 0.5 min, the column was re-equilibrated for 7.5 min. The injection volume was 2 μl.
For high resolution mass spectrometric detection and MS/MS experiments, a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ (Thermo Fisher Scientific, Waltham, MA, United States) mass spectrometer was operated in positive and negative electrospray ionization mode, respectively. A resolving power of 70,000 (FWHM @ m/z 200) was used to analyze the mass range m/z 113.4-1700.0. For data-dependent MS/MS experiments, the three most intense ions were fragmented with collision energy of 30 eV normalized at m/z 500. The instrument was controlled using the software Xcalibur™ 4.1 (Thermo Fisher Scientific™, Waltham, MA, United States). Unless stated otherwise in the figure description, the Q Exactive Plus was used for detection.
The data analysis of the high-resolution measurements was facilitated by the open-source software mzMine 2 (version 2.53; Pluskal et al., 2010). The accurate masses were detected with a noise level of 10,000 using the exact mass algorithm. For chromatogram building, the ADAP chromatogram builder was used (Myers et al., 2017). The minimum group size in number of scans was set to 4, the group intensity threshold to 50,000 and the minimum highest intensity to 10,000. The m/z tolerance was set to 0.001 m/z or to 15 ppm, whichever is higher. Savitzky-Golay smoothing with a filter width of 5 was applied, followed by chromatogram deconvolution using the wavelets (ADAP) algorithm. For deconvolution, the S/N was estimated using the intensity window and the S/N set to 3. The minimum feature height was set to 10,000 and the coefficient/area threshold set to 100. The retention time wavelet range was set from 0.00 to 0.15, and the median m/z center was calculated. Subsequently, isotopic peaks were grouped within an m/z tolerance of 0.001 m/z and 5 ppm, whichever is higher. The retention time tolerance was set to 0.1 min and the isotope with the lowest m/z was saved.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism version 8.4.2 software for Macintosh (GraphPad Software, La Jolla, CA, United States). To compare the outcome between different treatment groups and different animals the Mann-Whitney U test was used, while the two-tailed paired t test was used by comparing the contralateral knee joints within the same animal to determine the statistical significance of the collected data. All data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Intra-Articular Injection of Staphylococcus aureus LS-1 Strain Induces Bone Resorption
To study the effects of S. aureus on BMD in mice with septic arthritis, NMRI mice were i.a. injected with either S. aureus LS-1 strain, a pathogenic S. aureus strain that was originally isolated from a mouse that spontaneously developed septic arthritis (Bremell et al., 1991), or with PBS, and control NMRI mice were injected with PBS in both knees. Subsequently, a micro-CT evaluation was performed on the femoral BMD of mice with S. aureus septic arthritis. As expected, swelling in the joints infected with S. aureus started already on day 1 and continued until day 14 ( Figure 1A). Importantly, we observed that the i.a. injected S. aureus LS-1 strain significantly reduced total-and trabecular femoral BMD in mice when compared to contralateral knees injected with PBS, from 7 to 14 days post infection (Figures 1C,D). A trend toward BMD reduction, although not statistically significant was observed already on day 3 post infection (Figures 1C,D). These results imply that S. aureus-induced septic arthritis causes bone resorption in murine knee joints. Additionally, significant differences in totaland trabecular BMD were observed between the healthy mouse femoral bone and the femoral bone from PBS injected knee joints from the mice infected with S. aureus in their collateral knees (Figures 1C,D), suggesting that systemic inflammation caused by S. aureus local infection has significant impact on the bone metabolism. Representative imagines of the decrease in BMD, verified by micro-CT evaluation, were obtained on days 7 and 14 after i.a. injection with S. aureus ( Figure 1E). Similarly, S. aureus LS-1 i.a. injection significantly reduced total-and trabecular bone volume fraction (bone volume/tissue volume, Figures 1B,F), trabecular number ( Figure 1G), and trabecular thickness ( Figure 1H) on day 7 as well as day 14 post infection. At the early stage of the infection (on day 3), total-and trabecular bone volume fraction, trabecular number, as well as trabecular thickness were not significantly affected (Figures 1B,F-H). To assure our finding is not LS-1 strainspecific, another widely-used laboratory S. aureus Newman strain was injected to the mouse knee joints and BMD was analyzed on day 10 post infection. Both total and trabecular BMD were significantly downregulated in S. aureus Newman infected joints compared to healthy joints, suggesting that our finding is universal for different S. aureus strains (Supplementary Figure 1).

Intra-Articular Injection of Staphylococcus aureus Lpp Induces Bone Mineral Density Loss in a Lipid-Moiety Dependent Manner
Staphylococcus aureus Lpp is known to be one of the most potent bacterial components causing joint inflammation and bone destructions in septic arthritis (Mohammad et al., 2019). To further study whether the S. aureus Lpp induces loss of BMD, mice knee joints were i.a. injected with intact purified Lpl1(+sp) or PBS. Lpl1(+sp) is a model Lpp carrying the lipid moiety, which is derived from the νSaα-specific lipoproteinlike cluster (lpl) that exists in highly pathogenic and epidemic S. aureus strains (Nguyen et al., 2015). Significantly lower total-and trabecular BMD was observed in knees injected with Lpp compared to PBS on day 7 and 10 (Figures 2A,B). On the later time point (day 14 and 21) when the joint inflammation caused by Lpp was less pronounced, the difference of BMD in Lpp and PBS injected knees disappeared (Figures 2A,B). Representative images of the BMD loss caused by the S. aureus Lpp were obtained on day 7 post Lpp injection ( Figure 2C). To elucidate the molecular mechanism of BMD loss caused by the S. aureus Lpp, mice knee joints were i.a. injected with intact Lpl1(+sp) carrying the lipid moiety, or as control with Lpl1(−sp) which lacks the lipid-moiety. An analysis of the total-and trabecular BMD in femoral bone was subsequently performed 7 days after Lpp injection (Figures 2D,E). The mice injected with intact S. aureus Lpl1(+sp) displayed a significantly lower total-and trabecular BMD in comparison to the Lpl1(−sp) injected ones (Figures 2D,E). These results demonstrate that the lipid moiety in S. aureus Lpp is absolutely necessary for induction of bone loss.

Lpl1(+sp) Isolated From SA113 Contain Both Diacyl and Triacyl Lipid Moieties
To identify the lipid moiety structure, the tryptic Lpl1(+sp) was subjected to LC-MS analysis. As a result, two different tryptic lipopeptides can be detected; one cut directly after the first lysine (lipid-CGK), the other was cut after the second lysine (lipid-CGKGNETK) of the amino acid sequence of Lpl1. The detected short lipopeptides are mostly singly charged, the more abundant CGKGNETK lipopeptides can be detected as singly and doubly protonated molecules, with the doubly charged ones being more intensive. Both short and long peptides contain a mixture of lipid moieties with two-acyl and three-acyl residues, the latter carries in addition to the two diglyceride fatty acids also the N-acyl fatty acid. Supplementary Table 1 lists the lipopeptides identified using high resolution-MS. Assignments are based on chromatographic behavior and accurate mass (relative mass deviation less than 5 ppm compared to calculated theoretical mass). In addition, MS/MS experiments were performed for confirmation, using peptide fragments and neutral losses of fatty acids can be used to identify the lipopeptide structures.
The Diacylated Lipid-Moiety of the Synthetic Lipopeptide, Pam2Cys, Is More Potent in Inducing Bone Resorption Than the Triacylated Lipid-Moiety, Pam3Cys Since Lpl1(+sp) contained a mixture of diacyl and triacyl lipid moieties, we further compared the bone degenerative capabilities of the synthetic Pam2Cys and Pam3Cys lipopeptides that mimic the bacterial diacylated and triacylated Lpps respectively, on murine BMD. Mice knee joints were i.a. injected with 2 μg of either Pam2Cys or Pam3Cys. On day 7, we observed that Pam2Cys reduced the total-and trabecular BMD in mice significantly more than Pam3Cys (Figures 3A,B). As expected, the total BMD in femoral bone was significantly reduced by both i.a. injected Pam2Cys and Pam3Cys when compared to PBS i.a injected knees from healthy mice ( Figure 3A). Regarding the trabecular BMD, Pam3Cys tended to reduce the BMD (p = 0.08), whereas Pam2Cys caused a significant reduction when compared to the knee joints of control mice injected with PBS ( Figure 3B).

Monocytes, and Not Neutrophils, Are the Culprits Behind S. aureus Lpp-Induced Bone Destruction
Monocytes/macrophages, but not neutrophils, are known to be the immune cells responsible for the onset of S. aureus Lpp-induced arthritis (Mohammad et al., 2019). To further study the effect of monocyte/macrophage or neutrophil depletion on bone loss caused by Lpp, control mice or mice depleted of monocytes/macrophages by clodronate liposomes, or depleted of neutrophils by Ly6G antibodies, were i.a. injected into the knee with 10 μg of purified S. aureus Lpl1(+sp) or PBS. Injection of Lpl1(+sp) into knees in both control mice and neutrophil depleted mice caused a significantly lower total-and trabecular BMD in femoral bone than PBS (Figures 4A,B). Intriguingly, the depletion of monocytes/macrophages by clodronate liposomes gave rise to no significant differences in total BMD between Lpl1(+sp) and PBS i.a. injected knee joints 7 days post injection ( Figure 4A). However, a significant difference in trabecular BMD was observed between Lpl1(+sp) and PBS i.a. injected knee joints 7 days post injection ( Figure 4B). Our data suggest that monocytes/macrophages might be the main culprits causing BMD loss in Lpp injected knees. Significantly lower total-and trabecular BMD between Lpl1(+sp) of control mice compared to monocyte/macrophage depleted mice, and PBS of control mice compared to monocyte/ , with data expressed as the mean ± SEM (*p < 0.05, **p < 0.01, and ****p < 0.0001).

A B
FIGURE 3 | The diacylated lipid-moiety of the synthetic lipopeptide, Pam2Cys, is more potent in inducing bone resorption than the triacylated lipid-moiety, Pam3Cys. Graphs showing quantitative evaluations of (A) totaland (B) trabecular BMD (g/cm 3 ) of femoral mice bone (n = 8-12/group) on day 7 post i.a. knee injection with either Pam3Cys or Pam2Cys, or PBS collaterally in healthy control mice. Data were pooled from four independent experiments. Statistical evaluations were performed using the Paired t test between Pam2Cys and Pam3Cys injected knee joints (A,B) or Mann-Whitney U test between healthy control mice and Pam2Cys or Pam3Cys injected knee joints (A,B), with data expressed as the mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).
macrophage depleted mice, was observed on day 7 (Figures 4A,B), demonstrating the efficacy of clodronate in preventing bone absorption. Interestingly, total-and trabecular BMD tended to be lower in both Lpl1(+sp) and PBS injected knees from neutrophil depleted mice compared to control mice, indicating that neutrophils might be somehow bone protective (Figures 4A,B).
Monocytes and macrophages are one of the main producers of TNF-α and IL-1. Both cytokines are implicated in the development of osteoporosis (Kwan Tat et al., 2004). To further study the effects of IL-1 and TNF on bone absorption in Lpp-induced arthritis, S. aureus Lpp i.a. injected mice were treated with IL-1 or TNF inhibitors and BMD was subsequently analyzed and compared to control mice which received PBS. No significant differences in total-or trabecular BMD were observed between PBS and anti-IL1 or anti-TNF treated mice 10 days post i.a. injection with S. aureus Lpp (Figures 4C,D).

DISCUSSION
Tarkowski et al. demonstrated that hematogenous staphylococcal septic arthritis induces rapid systemic bone loss (Verdrengh et al., 2006). In the present study, we use a locally induced staphylococcal septic arthritis model to validate previous observations (Verdrengh et al., 2006), and further study the molecular and cellular mechanisms behind this effect. Our results demonstrate that local S. aureus septic arthritis causes systemic and local bone resorption. We found that the major players in bone resorption are the S. aureus Lpps that downregulate both total-and trabecular BMD. This effect is fully dependent on the lipid-moiety of Lpps. Interestingly, diacylated lipopeptides are more potent in inducing bone resorption than triacylated ones. Furthermore, monocyte depletion by i.v. injection of clodronate liposomes abrogates the osteoporotic effect of Lpps, suggesting that the recruited ) injection with 20 μl of purified S. aureus lipoprotein Lpl1(+sp; 10 μg/knee) or phosphate-buffered saline (PBS), measured in NMRI control mice (n = 6 knee joints/group) or NMRI mice depleted of either monocytes/macrophages using clodronate liposomes (n = 4 knee joints/group) or neutrophils using anti-Ly6G antibodies (n = 4 knee joints/group). (C) Total-and (D) trabecular BMD of femoral mouse knee joints 10 days post i.a. injection with 20 μl of purified Lpl1(+sp; 10 μg/knee), measured in NMRI mice treated with either anakinra, an interleukin 1 receptor antagonist (anti-IL-1) or etanercept, and a tumor necrosis factor inhibitor (anti-TNF) or PBS as control (n = 10 knee joints/group). Statistical evaluations were performed using the Paired t test between PBS and Lpl1(+sp) injected mice originating from the same host (A,B) or Mann-Whitney U test between PBS and Lpl1(+sp) injected mice of different treatment groups (A,B) or between PBS, anti-IL-1, or anti-TNF injected mice (C,D), with the data presented as a scatterplot with data expressed as the mean ± standard error of the mean (*p < 0.05 and **p < 0.01, ns = not significant).
Frontiers in Microbiology | www.frontiersin.org 8 March 2022 | Volume 13 | Article 843799 monocytes in local synovial tissue in septic arthritis may be the key mediators for systemic bone loss in septic arthritis. The binding of RANKL, a member of the TNF family, to its receptor, RANK, results in the fusion, differentiation, activation, and survival of osteoclasts (Lacey et al., 1998;Liu and Zhang, 2015). It has been demonstrated in previous ex vivo studies that S. aureus stimulation induces bone resorption and osteoclast differentiation by increasing osteoblast. Staphylococcus aureus induced RANKL expression in osteoblasts was entirely controlled by TLR2, because osteoblasts isolated from TLR2-deficient mice does not respond to S. aureus stimulation (Kassem et al., 2016). It is not surprising that Lpps are capable to induce the bone resorption, as TLR2 is known to be the ligand to S. aureus lipoproteins and Lpps are one of the primary arthritogenic S. aureus components (Mohammad et al., 2019). We observed that the lipid moiety in Lpp must present to reduce the murine BMD. The protein alone shows no significant activity.
Interestingly, we have found that Lpl1(+sp) isolated from SA113 clone contained a mixture of di-acyl and tri-acyl lipid moiety structures at N-terminal. Indeed, S. aureus has been shown to produce both di-acylated Lpp (Tawaratsumida et al., 2009) and tri-acylated Lpp (Kurokawa et al., 2009) under environmental conditions (Kurokawa et al., 2012). Recently, Gardiner and colleagues have discovered two genes, namely N-acylation transferase system A (lnsA) and B (lnsB) which were involved in N-acylation of Lpp in S. aureus (Gardiner et al., 2020). However, under which environmental conditions S. aureus produces more di-acylated Lpp or tri-acylated Lpp particularly in infection is not known. Since we demonstrate that both synthetic lipopeptides Pam2Cys and Pam3Cys significantly reduce murine BMD, we further investigated whether such lipopeptides can be produced in vivo. We digested purified Lpl1 with trypsin and found that a mixture of lipid-CGKGNETK and lipid-CGK were produced with di-or tri-acylations. This indicates that lipopeptides similar to the synthetic Pam2Cys and Pam3Cys can also be produced in infection.
Previous studies have demonstrated that the lipopeptides Pam2Cys and Pam3Cys induce osteoclast differentiation and activation by upregulating RANKL expression and suppressing osteoprotegerin expression in osteoblasts (Kim et al., 2013). In the current study, we have demonstrated that Pam2Cys displays increased potency compared to Pam3Cys in bone resorptive capacities, which is in line with previous findings by Nguyen et al. (2017), as Pam2Cys is known to be more potent in activating immune response and causing acute inflammation than Pam3Cys.
Although S. aureus Lpps have demonstrated a potent arthritogenic effect, it is very likely that other components of the bacteria may contribute to the development of osteoporosis in septic arthritis. For example, repetitive inhalation of peptidoglycan has been shown to induce bone loss in mice (Dusad et al., 2013). Furthermore, lipopolysaccharides and peptidoglycan have been reported to have a synergistic effect on bone resorption and osteoclastogenesis (Kishimoto et al., 2012). We infer that Lpps and peptidoglycan may also exhibit similar synergistic effects, as seen by Lpps in vitro stimulation boosting peptidoglycan stimulation of NOD2 (Schaffler et al., 2014). Our data suggest that the effect of BMD loss induced by S. aureus Lpps is of a complex time-dependent nature. On the later time point when joint inflammation caused by Lpp was less pronounced, the difference between PBS and Lpps injected knees disappeared. In contrast, in the case of live bacterial septic arthritis, the differences between PBS and bacteria injected knees were apparent on day 7 and continued to grow until day 14 post infection. We speculate that the discrepancy in time kinetics of BMD loss between Lpps and live S. aureus was mainly due to the different courses of disease models. In the bacterial septic arthritis setting the joint inflammation persists and accelerates after infection, which leads to continuous decline of BMD. In Lpps-induced arthritis setting, the differences in BMD become less apparent on the later time points when the inflammation was resolved.
Of note, the difference was also found in total and trabecular BMD between PBS and healthy knees on day 14 post infection when mice were infected intraarticularly with live S. aureus. This strongly suggests that systemic inflammation induced by local knee infection has significant impact on the osteoclastogenesis. The mice with septic arthritis were mobile and had only minimal weight loss at the beginning of the disease. However, they may avoid using the infected leg due to pain or discomfort. Immobility is known to result in rapid bone loss (Chan, 2005). Avoiding load bearing due to pain in the infected leg might be one of the contributing factors to the bone loss.
The strong interaction between bone and immune cells contributes to the pathogenesis of osteoporosis in humans (Fischer and Haffner-Luntzer, 2021). It is known that monocytes are circulating osteoclast precursor cells, and that bone erosions in bone diseases, such as rheumatoid arthritis (RA), may be caused by the differentiation of classical monocytes into osteoclasts (Rana et al., 2018). Our previous data demonstrated that i.a injection of Lpps induces rapid infiltration of monocytes into the synovial tissue (Mohammad et al., 2019). Mature monocytes and macrophages can be transformed to osteoclasts when a suitable microenvironment is provided by bone marrow-derived stromal cells (Udagawa et al., 1990). Based on our findings, we hypothesize that recruitment of monocytes to infected joints and transformation of monocytes/ macrophages to osteoclasts are the key steps of the rapid bone resorption in septic arthritis. However, further studies are warranted to elucidate detailed mechanisms in our animal models. Growth factors and cytokines such as macrophage colony-stimulating factor (M-CSF) and receptor activation of nuclear factor kappa-B ligand (RANKL) tightly regulate the activity and formation of osteoclasts. Both of these molecules are produced by synovial fibroblasts in the inflamed joints, and RANKL can also be produced by T-cells (Colucci et al., 2004) that are abundant in septic arthritis joints (Abdelnour et al., 1994). Moreover, differentiation of monocytes to osteoclasts can occur through stimulation with inflammatory cytokines such as TNF-α (Kim et al., 2005). Here, we also show that both Lpp and PBS injected mice with neutropenia exhibit lower BMD compared to control mice, which suggests that neutrophils exhibit a protective role in bone metabolism. In accordance with our results, it has been shown that patients with chronic neutropenia suffer from low BMD (Yakisan et al., 1997;Papadaki et al., 1999). In addition, the severity of the bone loss is strongly correlated with neutrophil counts (Papadaki et al., 1999). However, more studies are required to validate the results and further understand how the impact is mediated.
What is the clinical relevance of our findings? Advanced age and RA are risk factors for septic arthritis. Both RA and elderly patients are at a higher risk for developing osteoporosis. Our findings demonstrate that septic arthritis rapidly induces systemic BMD loss. To reduce the risk of bone fracture, we suggest examining all patients with a history of septic arthritis with bone densitometry, as osteoporosis is relatively easy to treat using bisphosphonate and RANKL inhibitors. Elderly and RA patients with a history of septic arthritis might have drastically increased risk for osteoporosis development and the anti-osteoporosis therapies could be introduced at the early stage of the disease. It has been demonstrated previously in a murine model that a combination of bisphosphonate, antibiotics, and anti-inflammatory treatment attenuates bone resorption in septic arthritis (Verdrengh et al., 2007), which suggests a new treatment modality for those patient groups.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author. Furthermore, the mass spectrometry data have been deposited to Proteome X change via the MassIVE partner repository with the data-set identifier MassIVE MSV000088739 (https://massive.ucsd.edu/ProteoSAFe/ static/massive.jsp). The data is provided as vendor specific .raw format and additionally as open .mzML format.

ETHICS STATEMENT
The animal study was reviewed and approved by The Ethics


Isospora basileuterusi n. sp. (Apicomplexa: Eimeriidae) from the golden-crowned warbler Basileuterus culicivorus (Deppe) (Passeriformes: Parulidae) in South America

Isospora basileuterusi Mello & Berto n. sp. is described based on material from the golden-crowned warbler Basileuterus culicivorus (Deppe) captured in the Itatiaia National Park (Parque Nacional do Itatiaia), a conservation unit in south-eastern Brazil. Oöcysts of the new species are ellipsoidal to ovoidal, measuring on average 25.2 × 21.1 μm, with a smooth, bi-layered wall, c.1.6 μm thick. Micropyle and oöcyst residuum are both absent, but one to three polar granules are present. Sporocysts are ellipsoidal to lemon-shaped, measuring on average 15.3 × 9.5 μm, with a knob-like Stieda body and a trapezoidal sub-Stieda body. Sporocyst residuum is present, usually as a body of membrane-bound granules. Sporozoites are vermiform, with refractile bodies. Four of the 19 warblers captured (21%) were infected with the new species. Molecular analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene revealed a similarity of 99.5% between the new species and Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries Serinus canaria (L.) in Western Australia. The oöcysts of I. basileuterusi n. sp. can be distinguished from the four other Isospora spp. recorded in hosts of the Parulidae, and from the molecularly most closely related species, by the typical ellipsoidal to lemon-shaped sporocysts, with small sub-Stieda body and a membrane-bound sporocyst residuum. Therefore, based on the morphological and molecular features, I. basileuterusi n. sp. is the fifth species described in a host of the family Parulidae and the first molecularly characterized via sequencing the cox1 gene.

Introduction
Brazil is the second country in the Neotropical region with the highest number of bird species, with about 1971 species listed by the Brazilian Ornithological Records Committee (Pacheco et al., 2021), which corresponds to half of the of the diversity of the Neotropical avifauna (Dias, 1992). In relation to research on Neotropical birds, the study of their parasites has been highlighted for their association with ecology, biology and species conservation. Among their parasites, coccidian protozoans are important as a cause of morbidity and mortality, especially in captive birds or impacted environments, thus acting as ecological biomarkers (Berto & Lopes, 2020).
The golden-crowned warbler Basileuterus culicivorus (Deppe) is a passerine bird of the family Parulidae with a wide distribution in the Neotropical region (Sick, 1997;Pacheco et al., 2021). It has insectivorous eating habits and occupies the middle stratum of the ombrophilous forests (Marini & Cavalcanti, 1993;Lima & Manhães, 2009). The present study provides a description and molecular characterization of a new species of Isospora from golden-crowned warblers B. culicivorus captured in the Itatiaia National Park (Parque Nacional do Itatiaia), a conservation unit in south-eastern Brazil.

Sample collection
A total of 9 expeditions were conducted between 2014 and 2019 in the Itatiaia National Park, a protected area with a high degree of vulnerability, located in the Serra da Mantiqueira on the border of the States of Rio de Janeiro, Minas Gerais and São Paulo (ICMBIO, 2021) . culicivorus were captured with mist nets. The birds were kept in individual boxes and faeces collected immediately after defecation. After identification to the species level, the bird was photographed and released, and stool samples were placed in centrifuge tubes containing 2.5% potassium dichromate (K 2 Cr 2 O 7 ) solution at 1:6 (v/v).

Morphological analyses
Samples were examined at the Laborat orio de Biologia de Coccídios, Universidade Federal Rural do Rio de Janeiro (UFRRJ). All samples were incubated at room temperature (25 C) for 10 days or until c.70% of the o€ ocysts were sporulated. O€ ocysts were isolated by flotation in Sheather's sugar saturated solution (specific gravity: 1.20) and examined microscopically using the technique described by Duszynski & Wilber (1997) and Berto et al. (2014a). Morphological observations, line drawings, photomicrographs and measurements were made using an Olympus BX binocular microscope (Olympus Optical, Tokyo, Japan) equipped with a digital camera Eurekam 5.0 (BEL Photonics, Monza, Italy). Line drawings were edited using two software applications (Corel DRAW and Corel PHOTO-PAINT) from CorelDRAW® (Corel Draw Graphics Suite, Version, 2020; Corel Corporation, Canada). All measurements are in micrometres and are given as the range followed by the mean in parentheses.

Molecular data generation
An individual o€ ocyst was isolated from serial dilutions of the o€ ocysts in drops on a microscope slide using a sterile micropipette. This isolated o€ ocyst was resuspended in PBS and washed by centrifuging until the supernatant became clear (Dolnik et al., 2009). DNA was extracted from the o€ ocyst using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, São Paulo, Brazil) according to the manufacturerʼs instructions. Four freeze-thaw cycles were applied prior to DNA extraction in order to achieve complete lysis of the o€ ocysts. PCR amplification of a partial fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene (c.250 bp) was carried out using nested PCR, as previously described by Dolnik et al. (2009) andYang et al. (2015). The external primers COIbF1 (5 0 -GWT CAT TAG TAT GGG CAC ATC A-3 0 ) and COIbR1 (5 0 -CCA AGA GAT AAT ACR AAR TGG AA-3 0 ), produced an amplicon of c.302 bp in size and the internal primes COIbF2 (5 0 -GGG CAC ATC ATA TGA C-3 0 ) and COIbR2 (5 0 -ATA GTA TGT ATC ATG TAR WGC AA-3 0 ) produced an amplicon of 257 bp in size. The PCR reaction contained 12.5 μl of GoTaq® G2 Hot Start Colorless Master Mix (Promega Labs, São Paulo, Brazil) (1 Â ), 0.25 μl of each primer (0.2 μM), 9 μl of nuclease-free water and 3 μl of DNA (for the primary reaction) or 3 μl primary PCR product (for the secondary reaction). Both primary and secondary PCR amplifications were conducted using the same cycling conditions: 1 cycle at 94 C for 5 min, followed by 35 cycles (94 C for 30 s, 47 C for 45 s, and 72 C for 1 min) and a final extension step at 72 C for 5 min. The amplicons from the second round PCRs were purified using the Qiagen MinElute PCR Purification (Qiagen, São Paulo, Brazil).

DNA sequence analyses
All PCR amplicons were sequenced using the PCR forward and reverse primers by Ludwig Biotechnology, where an ABI-Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, California) was used for Sanger sequencing. The results of the sequencing reactions were analyzed and edited using the program Chromas 2.6. The newly generated sequence was compared to those for Isospora spp. and other coccidian parasites available in the GenBank database using the Basic Local Alignment Search Tool (BLAST). The phylogenetic tree was constructed using the newly generated cox1 sequence aligned with sequences for 18 species of Isospora available on GenBank. Distance analyses and phylogenies were conducted using MEGA X (Kumar et al., 2018). Briefly, Sanger sequencing chromatogram files were imported into MEGA X and the nucleotide sequences were curated, analyzed, and aligned with reference sequences from GenBank using Clustal W (http://www. clustalw.genome.jp). Maximum likelihood (ML) and Neighbor-Joining (NJ) trees were constructed, and the distances computed using the Tamura-Nei method based on model selection using ModelTest in MEGA X. Bootstrap analyses were conducted using 1000 replicates to assess the reliability of inferred tree topologies.

Results
Nineteen B. culicivorus were examined and four (21%) were positive for coccidian o€ ocysts of a morphotype not reported in the scientific literature. These positive warblers were captured in November 2014 and August 2018 on a trail named "Trilha das Borboletas" (Trail of the Butterflies) (22 26 0 57 00 S, 44 36 0 25 00 W), and in March 2019 at the starting point of the "Travessia Ruy Braga" (Ruy Braga Crossing) (22 26 0 17 00 S; 44 37 0 33 00 W) in the Itatiaia National Park. This material is described below.
Site in host: Unknown. Prevalence: 21% (4 out of 19 birds examined). Representative DNA sequence: One representative cox1 sequence was deposited in the GenBank database under the accession number OM025014.
ZooBank registration: To comply with the regulations set out in Article 8. Etymology: The specific epithet is derived from the genus name of the type-host.

Discussion
Duszynski & Wilber (1997) compiled almost all taxonomic studies of coccidia of passerines and advised that new coccidian identifications should be based on comparative morphology between coccidian species recorded in the same host family. In this sense, the morphotype observed from the golden-crowned warblers in this study, I. basileuterusi n. sp., was compared with the four recorded coccidian species of Parulidae, as shown in Tables 1 and 2. Isospora basileuterusi n. sp. differs in several characteristic features, but can be mainly differentiated from the others by the typical lemon-shape of its sporocysts. The host of the new coccidian species described here, the goldencrowned warbler B. culicivorus, has a wide distribution in the Neotropical region, from Mexico to southern South America (Pacheco et al., 2021). However, according to BirdLife International (2021) this species is the stripe-crowned warbler, which is restricted to Mexico and Central America, not occurring in Brazil. This misinformation is due to species/subspecies status within the genus Basileuterus Cabanis. Birdlife have reclassified some subspecies of B. culicivorus to the species level, such as Basileuterus culicivorus auricapilla (Swainson, 1838) which has been reclassified to the level of species, as Basileuterus auricapilla Swainson, 1838. Therefore, this study followed the name listed by the Brazilian Ornithological Records Committee (Pacheco et al., 2021); however, it is noteworthy that the bird specimen in this study is identified as B. auricapilla by BirdLife International (2021). Anyway, regardless of the specific identification within Basileuterus, due to the wide geographical distributions of warblers (Parulidae) in the Americas, their coccidian species must be equally distributed throughout the Neotropical region.
In the present study, the molecular identification of I. basileuterusi n. sp. was performed using the cox1 gene, which is considered to be the gene with the highest resolution in detecting recent speciation events (Barta, 2001;Ogedengbe et al., 2011). In fact, the 250 bp cox1 gene sequence was not 100% similar to any other deposited on GenBank, contrary to what occurs with ribosomal gene sequences that are more conserved and more suitable for phylogenetic studies of families and orders . On the other hand, the region of the cox1 sequenced for I. basileuterusi n. sp. did not provide conclusive results related to ancestry, as linked to host family, biogeographical region, morphology/biology of the coccidian species, etc. (Fig. 3). This is also observed when Isospora spp. amplified and sequenced with the primers (Dolnik et al., 2009) used in the present study are exclusively included in the phylogeny, as in the studies of Yang et al. (2015) and Silva-Carvalho et al. (2018). Perhaps the short sequence of only 250 bp did not allow greater resolution in the phylogenetic study; in this case, sequences with more than 600 bp from other regions of the cox1 gene, such as those generated by the JAV primer , would show better phylogenetic estimations in the future. Fatally, in the present study these JAV primers were not successful in amplifying the samples; however, it has been shown in any case that mitochondrial genes, such as cox1, are better suited to work with individual o€ ocysts, as the number of copies of mitochondrial DNA is far greater than the number of copies of nuclear DNA, thus favoring the amplification of mitochondrial genes (Dolnik et al., 2009).

Conclusion
The comparison of I. basileuterusi n. sp. with Isospora spp. described from Neotropical warblers clearly supports the designation as a unique species. Therefore, I. basileuterusi is considered as new to science, which is the fifth species described in a host of the family Parulidae and the first molecularly characterized via sequencing the cox1 gene.

CRediT author statement
The study was designed by SVC, VML and BPB. Field work was performed by MSO, LASA and BPB. Laboratory procedures for maintenance, recovery, measurements, photomicrographs and isolation of o€ ocysts were performed by MSO and LASA. DNA extraction, amplification and sequencing were performed by ERM, AAO and VML. BPB analyzed the data and drew the coccidian o€ ocyst. The manuscript was written by ERM and BPB and subsequently revised by all other authors. All authors read and approved the final manuscript.

Data availability
Photosyntypes, line drawing, and o€ ocysts in 70% ethanol are deposited and available (http://r1.ufrrj.br/labicoc/colecao.html) in the Parasitology Collection of the Laborat orio de Biologia de Coccídios, at UFRRJ, under the repository number P-124/2021, along with the photographs of the type-host specimen (symbiotype). The generated sequence for I. basileuterusi n. sp. is deposited in the GenBank database under the accession number OM025014.

Declaration of competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.